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Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins.

ABSTRACT: The genomic region encoding the assembly protein of simian cytomegalovirus (CMV) strain Colburn has been cloned, sequenced, and found to be organized as a nested set of four in-frame, 3'-coterminal genes, each with its own TATA promoter element and translational start codon, and all using a single 3' polyadenylation signal. The 3' end of the longest open reading frame (1.770 bp) was identical to the 930-bp sequence coding for the assembly protein precursor, as determined from a cDNA clone. The assembly protein coding region of human CMV strain AD169 was similarly organized, suggesting that both viral genomes could give rise to four independently transcribed 3'-coterminal RNAs coding for four overlapping, in-frame, carboxy-coterminal proteins. These predictions were tested and confirmed. Four mRNAs corresponding in size and sequence to those predicted were identified in both human and simian CMV-infected cells by using transcript-specific antisense oligonucleotide probes in Northern (RNA blot) assays. The 5' ends of the three largest of these Colburn transcripts were determined by S1 nuclease protection assays and found to map between the anticipated TATA sequences and corresponding translational start codons. The four predicted overlapping proteins were identified by immunoassays in lysates of simian and human CMV-infected cells by using an antiserum specific for the carboxyl end of the assembly protein precursor. The structural relationship of both sets of proteins was verified by comparing their peptide patterns following protein cleavage at tryptophan residues by N-chlorosuccinimide. The similar organization of the homologous coding regions in other herpesviruses into at least two nested, in-frame, 3'-coterminal genes is discussed.

SUBMITTER: Welch AR

PROVIDER: S-EPMC248841 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins.

Welch A R AR McNally L M LM Gibson W W

Journal of virology 19910801 8

The genomic region encoding the assembly protein of simian cytomegalovirus (CMV) strain Colburn has been cloned, sequenced, and found to be organized as a nested set of four in-frame, 3'-coterminal genes, each with its own TATA promoter element and translational start codon, and all using a single 3' polyadenylation signal. The 3' end of the longest open reading frame (1.770 bp) was identical to the 930-bp sequence coding for the assembly protein precursor, as determined from a cDNA clone. The a ...[more]

PMID: 1649317

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Project description:Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

Project description:Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs.The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication.Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

Dataset Information

Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins.

Publications

Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins.

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